Regulation of epidermal homeostasis through P2Y2 receptors

*,1C. Jane Dixon, 1Wayne B. Bowler, 3Amanda Littlewood-Evans, 1Jane P. Dillon, 3Graeme Bilbe,2Graham R. Sharpe & 1James A. Gallagher

1Department of Human Anatomy and Cell Biology, University of Liverpool, New Medical School, Ashton Street, LiverpoolL69 3GE; 2Department of Dermatology, University of Liverpool, New Medical School, Ashton Street, Liverpool L69 3GE and3Novartis Pharma Inc. CH-4002 Basel, Switzerland

1 Previous studies have indicated a role for extracellular ATP in the regulation of epidermalhomeostasis. Here we have investigated the expression of P2Y2 receptors by human keratinocytes,the cells which comprise the epidermis.

2 Reverse transcriptase-polymerase chain reaction (RT±PCR) revealed expression of mRNA forthe G-protein-coupled, P2Y2 receptor in primary cultured human keratinocytes.

3 In situ hybridization studies of skin sections revealed that P2Y2 receptor transcripts wereexpressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y2

receptor transcripts to the basal layer of the epidermis, the site of cell proliferation.

4 Increases in intracellular free Ca2+ concentration ([Ca2+]i) in keratinocytes stimulated with ATPor UTP demonstrated the presence of functional P2Y receptors.

5 In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTPand ATPgS were found to stimulate the proliferation of keratinocytes.

6 Using a real-time ®re¯y luciferase and luciferin assay we have shown that under static conditionscultured human keratinocytes release ATP.

7 These ®ndings indicate that P2Y2 receptors play a major role in epidermal homeostasis, and mayprovide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis.

Keywords: Epidermis; keratinocytes; skin; proliferation; extracellular nucleotides; ATP; P2Y2 receptors; in situhybridization; intracellular calcium

Abbreviations: BrdU, bromodeoxyuridine; [Ca2+]i, intracellular free calcium concentration; cps, counts per second; DEPC,diethyl pyrocarbonate; GAPDH, glyceraldehyde phosphate dehydrogenase; Ins(1,4,5)P3, inositol(1,4,5)trisphos-phate; MAPK, mitogen-activated protein kinase

Introduction

Human epidermis is a strati®ed squamous epithelium

comprised of keratinocytes. Cell proliferation is largelycon®ned to the basal layer of the epidermis where cells divideto generate either replacement progenitor cells, or cells

committed to terminal di�erentiation. Di�erentiating cellsmigrate to the skin surface. The morphological andbiochemical changes which occur during di�erentiation

characterize the distinct layers of the epidermis (see Fuchs,1990). At the skin surface, the stratum corneum is composed ofdead, terminally-di�erentiated keratinocytes which form the

main cutaneous barrier. The balance between proliferation anddi�erentiation of basal cells, which maintains the epidermis atconstant thickness, is regulated by many extracellular factors;cell proliferation and di�erentiation usually follow an inverse

relationship. Positive growth factors include epidermal growthfactor (Rheinwald, 1980), transforming growth factor a(TGFa; Barrandon & Green, 1984; Gottlieb et al., 1988) and

keratinocyte growth factor (Finch et al., 1989). The bestcharacterized negative growth regulators, and hence stimula-tors of di�erentiation, are the TGFbs (Shipley et al., 1986),

extracellular Ca2+ (Hennings et al., 1980) and vitamin D(Jones & Sharpe, 1994).

Changes in keratinocyte intracellular free Ca2+ con-centration ([Ca2+]i) have been recorded in response to both

di�erentiation- and proliferation-stimulating agents,

although the pattern of change is dissimilar. Di�erentiation

is associated with a sustained increase in [Ca2+]i whichrequires in¯ux of extracellular Ca2+ to be maintained(Pillai et al., 1988, 1990; Bikle et al., 1996; Sharpe et al.,

1989). Recent evidence suggests that expression of theplasma membrane Ca2+-ATPase is decreased upon di�er-entiation, and that this contributes to the increased [Ca2+]i(Cho & Bikle, 1997). In contrast to this requirement for asustained increase in [Ca2+]i, growth stimulation isassociated with a temporary rise in [Ca2+]i lasting only a

few minutes, consistent with release of intracellular stores,but not Ca2+ in¯ux (Pillai & Bikle, 1992; McGovern etal., 1995).

In the vicinity of a wound, keratinocytes will be exposed

to ADP, ATP and UTP released from dead or damagedcells as well as from platelets (Boarder & Hourani, 1998).There is accumulating evidence that keratinocytes possess

speci®c receptors for these nucleotides, which may haveimportant e�ects on keratinocyte proliferation and di�er-entiation. Extracellular nucleotides such as ATP act at P2

receptors, a family of cell-surface receptors which have beendivided into two classes based on modes of signaltransduction and subsequently molecular cloning: theligand-gated ion channels, or P2X receptors; and G-

protein-coupled, P2Y receptors. There are a number ofsubdivisions of both classes that display di�erentialresponsiveness to nucleotide agonists (see Boarder &

Hourani, 1998); in the absence of speci®c antagonists thishas formed the basis of pharmacological characterization of*Author for correspondence; E-mail: C.J.Dixon@liv.ac.uk

British Journal of Pharmacology (1999) 127, 1680 ± 1686 ã 1999 Stockton Press All rights reserved 0007 ± 1188/99 $12.00

http://www.stockton-press.co.uk/bjp

receptor subtypes. All mammalian P2Y receptors cloned todate are coupled to hydrolysis of phosphatidylinositol 4,5-bisphosphate and hence to inositol 1,4,5-trisphosphate-

(Ins(1,4,5)P3-mediated release of intracellular Ca2+ stores(Boarder & Hourani, 1998). In addition P2Y2 receptoroccupation leads to activation of the mitogen-activatedprotein kinase (MAPK) cascade (see Boarder & Hourani,

1998); MAPK activation is closely associated with prolifera-tion.

The presence of P2Y receptors on primary human (Pillai &

Bikle, 1992) and canine keratinocytes (Suter et al., 1991) hasbeen indicated through recordings of increased [Ca2+]i inresponse to a range of extracellular nucleotides. The results of

desensitization experiments in both studies suggested that ATPand UTP were competing for a single receptor, indicating thatkeratinocytes express P2Y2 receptors. Consistent with the

reported transient nature of the ATP-evoked [Ca2+]i rise, thisnucleotide was also found to induce proliferation of humankeratinocytes and to inhibit di�erentiation (Pillai & Bikle,1992). However, in a separate study by Cook et al. (1995) ATP

was shown to have an inhibitory e�ect on human keratinocytegrowth. This anti-proliferative e�ect was not mediated byP2Y2 receptors, as UTP, an equipotent agonist at this receptor,

was ine�ective.To further understand the action of extracellular nucleo-

tides on keratinocytes we have investigated the presence and

function of P2Y2 receptors in human skin and culturedkeratinocytes. These ®ndings have important implications forthe actions of ATP and UTP in epidermal wound repair and

for the pathogenesis of hyperproliferative skin diseases. Wehave additionally demonstrated that under static conditionscultured human keratinocytes release ATP in signi®cantquantities. This indicates that keratinocytes in vivo may be

exposed to ATP, not only following injury, but also in thenormal skin micro-environment, raising the possibility thatATP has a role in epidermal homeostasis.

Methods

Cell culture

Normal human keratinocytes were isolated from foreskins or

neonatal rectoauricular skin removed during plastic surgeryand cultured in MCDB153 with supplements, as describedpreviously (Jones & Sharpe, 1994). Cells were passaged with the

addition of 100 mg ml71 bovine hypothalamic extract to aid cellattachment (Tsao et al., 1982) and used from passage 1 ± 4.

Isolation of RNA and ®rst strand cDNA synthesis

Total RNA was extracted from cultured keratinocytes or

tissue samples as described previously (Bowler et al., 1995),treated with RNAase-free DNAse 1 (35 U ml71) for 30 minsand stored as an ethanolic precipitate at 7208C. Analiquot of 5 mg of total RNA was used as template for ®rst

strand cDNA synthesis as described earlier (Bowler et al.,1995).

PCR analysis

PCR reactions were carried out using a 50 ml reaction volume

as described earlier (Bowler et al., 1995). Following an initialdenaturation step (948C for 2 mins), the conditions fordenaturation, annealing and extension were: 25, 30 or 35cycles of 948C for 10 s; 588C for 30 s (608C for glyceraldehyde

phosphate dehydrogenase (GAPDH); 728C for 30 s.Primer sequences:P2Y2: Sense 5'-CCAGGCCCCCGTGCTCTACTTTG

Anti-sense 5'-CATGTTGATGGCGTTGAGGGTGTGSense 5'-GGTGAAGGTCGGAGTCAACGG

GAPDH Anti-sense 5'-GGTCATGAGTCCTTCCACGAT

In situ hybridization

Sections of skin were prepared and stored according to the

protocol described earlier (Bowler et al., 1998). In situhybridization was performed as previously (Littlewood-Evans et al., 1997; Bowler et al., 1998). Brie¯y, sections

were rehydrated, treated with citric acid bu�er, andproteinase K to allow access of the RNA probes and todecrease non-speci®c binding. Sections were post-®xed in

paraformaldehyde to retain cellular localization of themRNA. Remaining proteins were acetylated with aceticanhydride. Prehybridization was carried out in a humidi®edbox at 428C for 3 h, before addition of 300,000 c.p.m. of

500 bp, sense or anti-sense 32P-dUTP-labelled P2Y2 ribo-probe per section. Hybridization was carried out overnightat 428C. Washing of the sections and development of slides

were conducted as described previously (Bowler et al.,1998). Finally, tissues were counter-stained in methyleneblue, mounted and photographed.

[Ca2+]i measurements

Keratinocytes were grown to approximately 70% con¯uencyon 22 mm diameter coverslips. The equipment and experi-mental protocol followed for the determination of [Ca2+]i havebeen described previously (Dixon et al., 1997). Measurements

were made from groups of approximately 8 ± 10 fura-2-loadedcells and addition of agonists was performed manually bypasteur pipette. Auto¯uoresence values were obtained in situ

using ionomycin and 50 mM manganese, as described byThomas & Delaville (1991). The ratio of ¯uorescenceintensities at 340 nm and 380 nm after subtraction of

auto¯uorescence was plotted as an indicator of [Ca2+]i.

Cell proliferation assay

The e�ect of extracellular nucleotides on proliferation ofkeratinocytes was assessed using a Biotrak cell proliferationELISA system (version 2). Brie¯y, keratinocytes were seeded at

subcon¯uent levels into 96 well plates and incubated inMCDB153 at 378C in a humidi®ed atmosphere of 5% CO2,95% air for 48 h. Medium was removed and replaced with

medium containing bromodeoxyuridine- (BrdU)-labellingreagent (1 : 1000) and the nucleotide of interest in the presenceof 10% FCS. Cells were incubated for a further 24 h before

®xing. Incorporation of BrdU was assessed by ELISA,according to the protocol of the manufacturer. Data wereanalysed by randomized block analysis of variance (ANOVA)followed by Newman Keuls procedure.

Real-time measurement of ATP release

ATP release was measured using a ®re¯y luciferase andluciferin assay in a custom-built photon-counting luminometerdescribed in detail in Cobbold & Lee (1991). A circular ¯at-

bottomed stainless steel chamber at 378C, with a capacity ofapproximately 300 ml was ®lled with HEPES bu�er ((mM)HEPES 10, NaCl 121, KCl 4.7, KH2PO4 1.2, MgSO4 1.2,CaCl2 2, NaHCO3 5, glucose 10, BSA 0.2%; pH 7.2)

P2Y2 receptors and skin 1681C.J. Dixon et al

containing 10 mg ml71 ®re¯y luciferase and 10 mM luciferin. A13 mm diameter coverslip of cells at con¯uency was ¯oatedgently into the full chamber to rest on its base. The chamber

was then sealed with a glass coverslip and positionedimmediately below the photocathode of the photomultiplier(EMI type 9789) housed in a constant-temperature incubator,held at 0 ± 48C to reduce electrical noise. The pulses of current

generated when light hit the photomultiplier were fed into acomputer ®tted with an EMI photon-counting board. Data,collected in 50 ms bins, were integrated over 1 s intervals to

give counts s71 (c.p.s.).The signals were calibrated by measuring the luciferase

luminescence over a range of ATP concentrations (1 nM ±

1 mM). The calibration curve, plotted as log of luminescenceintensity (c.p.s.) against log of [ATP], was linear over thisrange of concentrations.

Materials

FCS (Myoclone grade), Taq DNA polymerase and Super-

script II reverse transcriptase were purchased from GIBCOBRL, Paisley, U.K.; Fura-2 acetoxymethyl ester (fura-2 AM)was from Molecular Probes, Eugene, Oregon, U.S.A.; dNTPs,

oligo(dT), RNAase inhibitor and restriction enzymes wereobtained from Boehringer Mannheim, Lewes, U.K.; LM1emulsion and the Biotrak proliferation kit were from

Amersham, Little Chalfont, U.K.; MCDB153 culture medium,®re¯y luciferase and luciferin, and all other chemicals werepurchased from Sigma-Aldrich, Poole, U.K.

Results

Expression of P2Y2 receptor transcripts

cDNA derived from primary cultured human keratinocytes

was subjected to 25, 30 and 35 rounds of PCR ampli®cationusing oligonucleotide primers speci®c for the human P2Y2

receptor. The 362 bp product was clearly visible following 30

cycles of PCR ampli®cation demonstrating expression of P2Y2

receptor transcripts by this cell type (Figure 1A). We havepreviously demonstrated that human osteoclasts of giant celltumour abundantly express P2Y2 receptor mRNA (Bowler et

al., 1995). cDNA derived from a puri®ed population of humanosteoclasts was therefore included as a positive control; the362 bp product was visible following 25 cycles of ampli®cation

(Figure 1A). The speci®city of the ampli®ed product wascon®rmed by sequence analysis, and ampli®cation of GAPDHat 25 and 35 cycles con®rmed the integrity of cDNAs used

(Figure 1B).

P2Y2 receptor transcripts are localized to the stratumbasale

In situ hybridization revealed a striking pattern of localizationof P2Y2 receptor transcripts in the basal cell layer of skin

sections (Figure 2B, D and E). In control experiments with asense RNA probe no speci®c granular localization wasdetected (Figure 2A and C). Interestingly, high levels of

P2Y2 receptor mRNA were detected in both the sweat glands(Figure 2F) and the hair follicles (data not shown).

ATP and UTP increase [Ca2+]i in keratinocytes

Application of 10 mM ATP (n=5) or 10 mM UTP (n=5) togroups of 8 ± 10 fura-2 loaded keratinocytes induced a rise in

[Ca2+]i. Figure 3 depicts the response of a group ofkeratinocytes to increasing concentrations of ATP and UTP.The threshold concentrations for ATP and UTP were in the

low micromolar range; 1 mM UTP evoked a response in 2/4preparations, and 2 mM ATP was e�ective in 3/4 prepara-tions (1 mM ATP did not elicit a rise in [Ca2+]i in 4/4preparations).

P2Y2 agonists stimulate proliferation of humankeratinocytes

Keratinocytes cultured in MCDB153, supplemented asdescribed above, are undergoing near maximal proliferation.

In order then to study the e�ect of nucleotide stimulation therate of proliferation needs to be reduced. This was achieved byaddition of 10% FCS, which slows keratinocyte growth as

noted previously (Sharpe & Fisher, 1990). In the presence of10% FCS, ATP (0.1 ± 100 mM) signi®cantly increased prolif-eration (Figure 4A). The poorly-hydrolyzable analogue ofATP, ATPgS, and UTP similarly stimulated keratinocyte

proliferation in the presence of serum (Figure 4B).

Keratinocytes constitutively release ATP

As depicted in Figure 5, introducing a coverslip of con¯uentkeratinocytes into the chamber of the photon-counting

detection system led to an immediate increase in the intensityof the luminescence. The signal rose from the background levelof approximately 9 c.p.s. to 604+166 c.p.s. (mean+s.e.mean,

A

B

Figure 1 Expression of P2Y2 receptor transcripts in primarycultured human keratinocytes and osteoclasts. (A) PCR ampli®cationof a speci®c 362 bp fragment of the human P2Y2 receptor usingcDNA from primary cultured human keratinocytes. cDNA from asorted human osteoclast population was included as a positivecontrol. (B) Ampli®cation of a speci®c 519 bp GAPDH productcon®rmed the integrity of the cDNA used. This result is typical ofthree analyses using keratinocytes derived from skin samples fromthree individual patients.

P2Y2 receptors and skin1682 C.J. Dixon et al

C D

E F

A B

Figure 2 In situ localization of P2Y2 receptor transcripts in sections of human skin using 32P-dUTP-labelled probes. (A) Lowpower photomicrograph of hybridization with a sense probe (negative control) showing no speci®c granular localization. (B) Similarsection hybridized with an anti-sense probe and exposed for 15 days. Note the localization over the basal (proliferative) layer of theepidermis and in the glandular structures deep in the dermis. (C) High magni®cation view of a skin section hybridized with a senseprobe (negative control) and exposed for 5 days. Note the absence of signal. (D and E) High magni®cation detail of skin sectionshybridized with anti-sense probe and exposed for 5 days and 15 days respectively. Note the intense speci®c localization of signalover the basal (proliferative) layer of the epidermis. (F) High magni®cation view showing speci®c hybridization of the anti-senseprobe over sweat glands.

P2Y2 receptors and skin 1683C.J. Dixon et al

n=5). This equates to a concentration of ATP in the chamberof 17+4 nM, although considerable variability in the level of

ATP release from individual coverslips was seen (range: 7 ±30 nM). The plateau in signal was achieved when the rate ofrelease of ATP was equal to the rate of its removal throughhydrolysis and the action of extracellular enzymes. The signal

remained steady at this level for the length of the recording; no

change was seen even over a period of 50 min. If extreme care

was not taken when introducing keratinocytes into thechamber, a large increase in signal was recorded, which couldpeak at 2000 ± 3000 c.p.s., and which decayed gradually over a

period of 2 ± 3 h (data not shown). This signal may re¯ect lysisof some cells with concomitant release of intracellular ATP.Alternatively, it may represent the physiological response of

cells to the mechanical stresses generated when a coverslip isintroduced into the chamber.

Discussion

In these studies we have described the expression of P2Y2

receptor mRNA transcripts in cultured human keratinocytes.P2Y2 receptor expression has been shown to be up-regulated inresponse to culture conditions (Turner et al., 1997). However,

in situ hybridization studies presented here clearly demonstratethat transcripts for these receptors are present in the nativetissue. These studies illustrate a striking pattern of localizationof transcripts to the actively-proliferating basal cells of the

epidermis. P2Y2 receptor transcripts were also detected in theouter, more di�erentiated layers of the epidermis, although atmuch lower levels. This suggests that P2Y2 mRNA receptor

expression is down-regulated in a di�erentiation-dependentmanner, and is therefore reminiscent of the P2Y2 receptormRNA down-regulation observed during phorbol ester-

induced monocytic di�erentiation of HL60 human promyelo-cytic leukocytes (Dubyak et al., 1996; Martin et al., 1997).

The most common primary e�ectors of G-protein-coupled

P2Y receptors are the phospholipase C b enzymes; activationof receptors leads to Ins(1,4,5)P3-mediated release ofintracellular Ca2+ stores. In keeping with an earlier study onhuman keratinocytes (Pillai & Bikle, 1992) stimulation with

ATP and UTP, equipotent agonists for the P2Y2 receptor,resulted in increased [Ca2+]i. This suggests that the detectedtranscripts are translated into functional receptors, although a

contribution from other receptors (e.g. P2Y4 and P2Y11) to theresponses to ATP and UTP cannot be excluded.

The intense localization of P2Y2 mRNA transcripts to the

actively-dividing basal layer suggests a role for nucleotides as

Figure 3 Increases in [Ca2+]i induced by ATP and UTP in culturedhuman keratinocytes. Cultured keratinocytes were loaded with fura-2by incubation with 5 mM fura-2 AM. Groups of 8 ± 10 cells wereexcited with light at 340 and 380 nm and the emission at 510 nmrecorded. The application of ATP or UTP led to increases in [Ca2+]ias indicated by the change in the ratio of ¯uorescence at 340 and380 nm.

A

B

Figure 4 Proliferation of keratinocytes in response to (A) increasingconcentrations of ATP and (B) 100 nM ATP, UTP and ATPgS wasassessed using a Biotrak cell proliferation ELISA system. All data arerepresented as mean+s.e.mean (n=16 wells). * denotes signi®cantdi�erence at P50.01. This result is typical of four separateexperiments.

Figure 5 ATP release from human keratinocytes. The chamber ofthe photon-counting luminometer was ®lled with ®re¯y luciferase(10 mg ml71) and luciferin (10 mM) in HEPES bu�er and thebackground signal recorded. Introduction of a coverslip ofkeratinocytes at con¯uency resulted in an increase in signal toapproximately 500 c.p.s. The signal was calibrated by measuring theluciferase luminescence over a range of ATP concentrations as shownon the right hand axis. This result is typical of ®ve coverslipsalthough considerable variability was seen in the signals recordedfrom individual coverslips.

P2Y2 receptors and skin1684 C.J. Dixon et al

regulators of epidermal proliferation. Keratinocytes cultured inMCDB153 medium, supplemented as described above, pro-liferate at near maximal rate and further stimulation of the

growth rate will not readily be observed. In order then to studythe e�ect of nucleotide stimulation on keratinocyte growth, therate of proliferation must ®rst be slowed. This was achieved bythe addition of 10% FCS, which slows proliferation and

promotes di�erentiation of cells. The inhibitory e�ect of serumhas been noted previously and has been attributed, at least inpart, to the high concentrations of TGFb it contains (Sharpe &

Fisher, 1990). Under these conditions then, stimulation withATP and UTP led to a signi®cant increase in proliferation, thusover-riding the inhibitory e�ects of factors present in serum.

However, this response to ATP appears to be independent of[Ca2+]i, as proliferation was seen at concentrations lower thanthe threshold for [Ca2+]i mobilization. This may re¯ect

involvement of alternative signalling pathways in mediatingthe ATP-stimulated proliferation. Indeed P2Y2 occupation hasrecently been shown to lead to activation of the p42/p44MAPK cascade which is associated with proliferation (Boarder

&Hourani, 1998). Mitogenic e�ects of ATP have been reportedpreviously in a number of cell types including aortic smoothmuscle cells (Wang et al., 1992; Harper et al., 1998), mesanglial

cells (Schulze-Loho� et al., 1992), Swiss 3T3 and 3T6®broblasts (Huang et al., 1989) and signi®cantly in primaryhuman keratinocytes (Pillai & Bikle, 1992).

In vivo, keratinocytes may be exposed to high levels of ATPand UTP following injury to the skin when nucleotides arereleased from dead or damaged tissue, and from platelets. The

resulting proliferation of cells in the basal layer would promotehealing of an epidermal wound by replacing damagedepidermis. Growth factors, including TGFa which is knownto stimulate keratinocyte proliferation, are released along with

nucleotides upon degranulation of platelets at the site of aninjury. ATP has been shown to interact synergistically withgrowth factors to promote cell proliferation in a range of cell

types including a mouse keratinocyte-derived cell line, BALB/MK (Wang et al., 1990).

In partial epidermal loss, proliferation of the basal

keratinocytes re-forms the full epidermis. In deeper injury,new epidermal growth occurs from the hair follicles wherepreserved, or from the wound edges. The dense localization ofP2Y2 mRNA transcripts to the hair follicles reported here may

explain the ability of the skin to regenerate from these regions;extracellular nucleotides acting at the P2Y2 receptors wouldstimulate proliferation of basal cells within the inner root

sheath of the follicle. High levels of P2Y2 mRNA were alsodetected in the sweat glands, consistent with an established rolefor these receptors in regulating ion conductance into sweat

(Ko et al., 1997; Wilson et al., 1996).Luciferase/luciferin experiments demonstrate that under

static conditions keratinocytes are releasing ATP. The amount

of ATP released into the 300 ml volume of the chamber gaverise to an ATP concentration of approximately 17 nM.Released into a restricted volume at the cell surface, ATPmay reach su�cient concentrations to activate P2Y2 receptors

and hence stimulate proliferation of keratinocytes. Thestimulation of ATP release by extracellular ATP has beendemonstrated in endothelial cells (Bodin & Burnstock, 1996),

and thus the basal release recorded here may re¯ect ATP-induced ATP release. Interestingly, serum contains highconcentrations of enzymes which hydrolyze ATP (Suter et al.,1991). If released ATP contributes to the proliferation of

keratinocytes in culture, the inhibitory e�ects of serum onproliferation may be partly explained by the removal of ATP,and hence the proliferative stimulus.

Release of ATP has been demonstrated from a number ofcell types in response to mechanical stimulation (Milner et al.,1990; Schlosser et al., 1996; Grygorczyk & Hanrahan, 1997).

Indeed, the large signal observed when introducing a coverslipinto the chamber without exercising extreme care may re¯ectthe response of keratinocytes to mechanical stress. In skin,

mechanical forces of pressure and shearing are transmitted tothe basal layer and basement membrane (Kennedy, 1998).Repeated shearing forces acting on an area of skin results inthickening of the area. This physiological response may be

mediated by ATP, released in response to shearing forces.Recently, release of UTP has been demonstrated from 1321N1astrocytoma cells (Lazarowski et al., 1997). If UTP is similarly

released from keratinocytes in response to mechanicalstimulation, this nucleotide may act in concert with ATP toregulate proliferation.

It is likely that keratinocytes express other P2Y subtypes inaddition to the P2Y2 receptor, possibly also in a di�erentia-tion-dependent manner. Indeed, Pillai & Bikle (1992) reported

that cultured keratinocytes respond to ADP with increased[Ca2+]i; this nucleotide is not an agonist for the P2Y2 receptor,but acts at both the P2Y1 and P2Y6 subtypes (see Boarder &Hourani, 1998). Further expression and functional studies are

required to determine the full complement of P2 receptorsexpressed by keratinocytes.

In conclusion, we have described the expression and

involvement of P2Y2 receptors in the control of keratinocyteproliferation. ATP and UTP, released in the vicinity of awound from damaged cells or as a result of the in¯ammatory

response, may act to promote keratinocyte proliferation andinhibit di�erentiation through activation of P2Y2 receptors.This mechanism may be responsible for epidermal thickeningdue to repeated minor trauma or shearing forces if, as seems

likely, keratinocytes release ATP (and possibly also UTP) inresponse to mechanical stimulation. Agonists for the P2Y2

receptor thus represent potential therapeutic agents for the

enhancement of wound healing, whilst antagonists may beuseful in the treatment of pathologies associated withhyperproliferation of keratinocytes, including psoriasis. In

addition, the signi®cant concentrations of ATP released bykeratinocytes under static conditions, indicate that ATP mayact as an important positive growth regulator.

We are grateful for funding from The Wellcome Trust (C.J. Dixon)and the Arthritis and Rheumatism Research Council (W.B. Bowler).

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(Received November 20, 1998Revised March 19, 1999Accepted April 14, 1999)

P2Y2 receptors and skin1686 C.J. Dixon et al